MINISTRY OF EDUCATION AND TRAINING VINH UNIVERSITY VU THI HIEN STUDY ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITY OF FRUITS OF Aphanamixis polystachya Wall.. For that reason, we h
Trang 1MINISTRY OF EDUCATION AND TRAINING
VINH UNIVERSITY
VU THI HIEN
STUDY ON CHEMICAL CONSTITUENTS
AND BIOLOGICAL ACTIVITY OF FRUITS OF
Aphanamixis polystachya (Wall.) R.Parker, Khaya senegalensis A Juss AND Melia azedarach L
BELONGING TO THE FAMILY MELIACEAE IN
Trang 2The present study has been completed at: Specialized Lab Organic
Chemistry, Vinh University
Supervisors:
1 Assoc Prof Dr Vu Dinh Hoang
2 Assoc Prof Dr Hoang Van Luu
The thesis will be defended at the High-tech buildings at Vinh University, 182 Le Duan, Vinh City, Nghe An Province, 2019
At 8 h 00
The thesis can be found at:
1 The National Library of Vietnam
2 Centre for information - Library Nguyen Thuc Hao, Vinh
University
Trang 3Species Aphanamixis polystachya, Khaya senegalensis and Melia azedarach
belong to Vietnamese folk medicinal plants However, until now, almost no research
on the chemical composition and biological activity of these plants in Vietnam has been reported This thesis is aimed to clarify the use in folk, improve the effectiveness and safety of medicinal plants, develop medicinal materials to use in a more scientific way Therefore, the study field has important theoretical and practical significance, making an important contribution to finding natural resources, economic value of Vietnam's natural medicinal resources For that reason, we have
chosen the topic: "Study on chemical composition and biological activity of
fruits of Anphanamixis polystachya, Khaya senegalensis and Melia azedarach
belonging to family Meliaceae in Vietnam”
2 Objects
The research objects of the thesis are extracts from fruits of Anphanamixis
3 Tasks
The research tasks include:
- Selecting suitable solvents to extract the existing compounds from fruits of
Vietnam
- Separating and identifying structures of the compounds isolated
- Assaying biological activities of the concerned compounds
4 Methods
- Collection samples: collection, identification, cultivation for some fungi, storage and preservation dried fungi of 2 samples at the laboratory temperature (20-300C)
- Isolation of compounds by a combination of chromatographic techniques; column chromatography (CC), thin layer chromatography (TLC) with different
Trang 4stationary phases are silica gel, Sephadex LH-20, RP-18; high-performance liquid chromatography (HPLC)
- Structure elucidation by UV, IR, mass spectrometry (MS), as well as one and two dimensional NMR techniques (1H-NMR, 13C-NMR, 2D- 1H-1H-COSY, HMBC, HSQC)
- Test of anti-inflammatory and antibacterial activities and plant pathogenic antifungal activity of pure compounds
5 New contributions of the thesis
Study the chemical composition and biological activity of fruits of Anphanamixis polystachya, Khaya senegalensis, Melia azedarach in Vietnam, we have
obtained some results as follows:
1 Isolated and determined the structure 6 compounds from fruits extract of
Aphanamixis polystachya, include:
compound G và 6α - acetoxyepoxyazadiradione VI
- 02 sterol compounds: β-sitosterol, -sitosterol-3-O--D-glucopyranoside dysobinin, chisocheton compound E, chisocheton compound G và 6α - acetoxyepoxyazadiradione VIwere isolated for the first time
6-acetoxy-methyl angolensate
- 02 flavonoid compounds: (-)-epicatechin and quercitrin
3 Isolated and determined the structure 8 compounds from fruits extract of Melia
3α,12α-diacetoxy-7α-benzoyloxy-1α-hydroxytrichilinin which is called trichilinin F
- 02 flavonoid compounds: apigenin and quercetin 3-O-[(1→6)]--D-glucopyranoside)
-L-rhamnopyranosyl 01 triterpenoid compound: taraxerol
- 02 phenolic compounds: scopoletin and acid vanillic
- 02 sterol compounds: β-sitosterol, -sitosterol-3-O--D-glucopyranoside
4 Test of anti-inflammatory biological activity and antifungal activity that causes
plant diseases Evaluate the antifungal activity of compounds isolated with C
obscurans
6 Outline of the thesis
Trang 5It is displayed in a total of 112 pages with 21 tables, 19 figures, 3 diagrams Its major sections include: Introduction (4 pages), overview (25 pages), methods and experiment (20 pages), results and discussion (69 pages), conclusion (1 pages), published works (1 page) Morever, there is an appendix with 130 spectra
2 Species Aphanamixis polystachya
- Introduction of botanical characteristics of Aphanamixis polystachya
- The chemical composition of Aphanamixis polystachya
- The biological activity of Aphanamixis polystachya
3 Species Khaya senegalensis
- Introduction of botanical characteristics of Khaya senegalensis
- The chemical composition of Khaya senegalensis
- The biological activity of Khaya senegalensis
4 Species Khaya senegalensis
- Introduction of botanical characteristics of Melia azedarach
- The chemical composition of Melia azedarach
- Biological activity of Melia azedarach
CHAPTER 2: METHODS AND EXPERIMENT
2.1 Methods
2.1.1 Method of collecting samples
The plant material is collected at the appropriate time of the year When fresh plant material is required for study, they are washed, stored in cool place, isolation, preservation, and fermentation They are stored at appropriate conditions for the experiment
Trang 62.1.2 Methods of separation
Thin layer chromatography (TLC); column chromatography (CC); lash column chromatography (FC); high performance liquid chromatography (HPLC); fractional crystallization
2.1.3 Methods of identifying the component structure
Using modern spectroscopic methods to demonstrate structure of obtained compound consisting of ultraviolet (UV), infrared (IR), mass spectrometry (ESI-
MS, HR-ESI-MS), nuclear magnetic resonance spectroscopic 1H-NMR, 13NMR, DEPT and HSQC, HMBC, 1H-1H COSY
C-2.1.4 Methods of bioactivity assay
Test anti-inflammatory activity and plant anti-fungal activity Evaluate the
gloeosporioides, F oxysporum, B cinerea và P obscurans
2.2 Chemicals and equipment
2.2.1 Chemicals: Solvents for extraction plant material are pure The solvents
for thin layer chromatography, fast column chromatography are pure analytical
(PA)
2.2.2 Equipment: Melting temperature was determined with a Yanaco MP-S3;
The polarity was determined with a Jasco DIP -370 polarimeter Thin layer
chromatography (TLC); column chromatography (CC); lash column
chromatography (FC); high performance liquid chromatography (HPLC); ultraviolet (UV), infrared (IR), mass spectrometry (ESI-MS, HR-ESI-MS), nuclear magnetic resonance spectroscopic (NMR), optical rotation and melting points measurement;
2.3 Study on compounds from fruits of Aphanamixis polystachya
2.3.1 Collecting samples
The fruits of Anphanamixis polystachya was collected at the Vu Quang
National Park of Ha Tinh Province, Vietnam, in August 2013 and identified by Assoc Prof Dr Tran Huy Thai, Institute of Ecology and Biological Resources - Vietnam Academy of Science and Technology A voucher specimen (DHV-2013) was deposited at the herbarium of the Department of Chemistry, Vinh University
2.3.2 Isolating substances
The fruits of Anphanamixis polystachya (4.0 kg) were air-dried and
powdered and soaked with methanol at room temperature for 7 days, and the combined extracts were concentrated under reduced pressure to give deep brown syrup (290.0 g) The crude extract was suspended into water and partitioned with cloroform and butanol, successively to afford cloroform (106 g), butanol (35.0 g), respectively, after removal of the corresponding solvent
Trang 7The chloroform soluble extracts were purified by silica gel column chromatography eluted with n-hexane and acetone gradients (100:0, 25:1, 15:1, 10:1, 7:1, 5:1) and CHCl3:CH3OH (100:0, 6:1, 3:1, 2:1, 1:1) gradients to afford 7 fractions (F1 to F7)
Fraction F1 (8.6 g) was subjected to silica gel column chromatography eluted (200 gam, 60 x 5 cm) with n-hexane/acetone (100:0, 25:1, 15:1, 10:1, 4:1)
to afford seven subfractions (F1-1 to F1-7) Subfraction F1-4 was subjected to the silica gel column chromatography eluted with n-hexane/acetone (100:0, 25:1,
15:1, 10:1, 4:1) to afford AP-5 (153 mg)
Fraction F2 (2.3 g) was subjected to silica gel column chromatography eluted (200 gam, 60 x 3 cm) with n-hexane/acetone (9:1, 6:1) to afford six subfractions (F2-1 to F2-6) Subfraction F2-6 was subjected to the Sephadex LH-
20 column chromatography eluted (50 gam, 60x3cm) with CH3OH:H2O to afford
AP-1 (41 mg)
Fraction F3 (2.7 g) was subjected to silica gel column chromatography eluted (200 gam, 60 x 3 cm) with n-hexane/acetone (9:1, 6:1, 4:1, 1:1) to afford four subfractions (F3-1 to F3-4) Subfraction F3-2 was subjected to the phase reversal (RP -18 ) column chromatography eluted (100 gam, 60 x 3 cm) with
CH3OH:H2O to afford AP-2 (31 mg)
Fraction F4 (4.7 g) was subjected to silica gel column chromatography eluted (200 gam, 60 x 3 cm) with CHCl3:CH3OH (20:1, 10:1, 6:1, 4:1, 2:1) to afford five subfractions (F4-1 to F3-5) Subfraction F4-1 was subjected to the phase reversal (RP -18 ) column chromatography eluted (100 gam, 60 x 3 cm) with CH3OH:H2O (19:1, 16:1) to afford AP-4 (43 mg) and AP-3 (21 mg)
Fraction F5 (1.5 g) was subjected to silica gel column chromatography eluted (200 gam, 60 x 3 cm) with CHCl3:CH3OH (9:1, 6:1) to afford AP-6 (13
mg)
2.4 Study on chemical constituents of fruits of Khaya senegalensis A Juss
2.4.1 Collecting samples
The fruits of Khaya senegalensis A Juss was collected at the Nghe An
Province, Vietnam, in January 2014 and identified by Assoc Prof Dr Tran Huy Thai, Institute of Ecology and Biological Resources - Vietnam Academy of Science and Technology A voucher specimen was deposited at the herbarium of the Department of Chemistry, Vinh University
2.4.2 Isolating substances
The fruits of Anphanamixis polystachya (4.0 kg) were air-dried and
powdered and soaked with methanol at room temperature for 7 days, and the combined extracts were concentrated under reduced pressure to give deep brown
Trang 8syrup (285.0 g) The crude extract was suspended into water and partitioned with n-hexane and ethyl acetate, successively to afford n-hexane (55g), butanol (95.0 g), respectively, after removal of the corresponding solvent
The ethyl acetate soluble extracts were purified by silica gel column chromatography eluted with n-hexane and acetone gradients (100:0; 50:1; 39:1; 30:1; 20:1; 15:1; 9:1; 4:1; 2:1; 1:1) to afford 7 fractions (F1 to F7)
Fraction F3 was subjected to silica gel column chromatography eluted with n-hexane/acetone (20:1; 15:1; 9:1; 2:1) to afford five subfractions (F3-1 to F3-5) Subfraction F13-3 was subjected to the silica gel column chromatography eluted
with n-hexane/acetone (9:1; 4:1) to afford KS1 (15 mg) and KS3 (29 mg)
Fraction F4 was subjected to silica gel column chromatography eluted with
chloroform:methanol (9:1) to afford KS4 (12.5 mg)
Fraction F5 was subjected to silica gel column chromatography eluted with n-hexane/acetone (15:1; 9:1; 4:1; 2:1) to afford seven subfractions (F5-1 to F5-7) Subfraction F5-2 was subjected to silica gel column chromatography eluted to
afford KS5 (53 mg)
Fraction F5-4 (4.7 g) was subjected to silica gel column chromatography
eluted with chloroform: methanol (15:1, 9:1) to afford KS6 (71.5 mg).
Trang 92.5 Study on chemical constituents of fruits of Melia azedarach
2.5.1 Collecting samples
The fruits of Melia azedarach was collected at the Pu Huong National Park
of Nghe An Province, Vietnam, in August 2016 and identified by Assoc Prof
Dr Tran Huy Thai, Institute of Ecology and Biological Resources - Vietnam Academy of Science and Technology A voucher specimen (DHV 2016) was deposited at the herbarium of the Department of Chemistry, Vinh University
2.5.2 Isolating substances
The fruits of Anphanamixis polystachya (5.0 kg) were air-dried and
powdered and soaked with methanol at room temperature for 7 days, and the combined extracts were concentrated under reduced pressure to give deep brown syrup (425.0 g) The crude extract was suspended into water and partitioned with ethyl acetate and n-butanol, successively to afford chloroform (106 g), n-butanol (35.0 g), respectively, after removal of the corresponding solvent
The ethyl acetate soluble extract was fractionated by silica gel column chromatography eluted with chloroform and methanol gradients (100:0, 40:1: 30:1; 20:1; 10:1: 4:1; 2:1) to afford 7 fractions (F1 to F7)
Fraction F1 was subjected to silica gel column chromatography eluted with
n-hexane/acetone (15:1) to afford MA6 (128 mg)
Fraction F3 was subjected to silica gel column chromatography eluted with
n-hexane/acetone (7:1) to afford MA3 (28 mg) and MA5 (31 mg)
Fraction F5 was subjected to silica gel column chromatography eluted with
chloroform: methanol (10:1) to afford MA4 (51 mg)
The n-butanol soluble extract was fractionated by silica gel column chromatography eluted with chloroform and methanol gradients (30:1; 20:1; 10:1: 4:1; 2:1) to afford 5 fractions Fraction 2 was subjected to the silica gel
column chromatography eluted with chloroform: methanol (15:1) to afford MA1
(12 mg) Fraction 3 was subjected to the silica gel column chromatography
eluted with chloroform: methanol (10:1) to afford MA2 (21.5 mg) Fraction 5
was subjected to the silica gel column chromatography eluted with chloroform:
methanol (10:1) to afford MA7 (41 mg)
Trang 102.6 Biological activity test
Test anti-inflammatory activity and plant anti-fungal activity Evaluate the
antifungal activity of compounds isolated with C acutatum, C fragariae, C
gloeosporioides, F oxysporum, B cinerea và P obscurans
CHAPTER 3: RESULTS AND DISCUSSION
3.1 Fruits of Anphanamixis polystachya
Table 3.1: Isolated compounds from fruits of Anphanamixis polystachya
3.2 Fruits of Khaya senegalensis
Table 3.2: Isolated compounds from fruits of Khaya senegalensis
The process of isolating the substances from the fruits of Melia azedarach
was presented in detail in the experimental section In the course of our research
we have focused on studying the chemical composition of the methanol extract from leaves Annona reticulata by combining the methods of extraction, thin layer
Trang 11chromatography, silica gel column chromatography, high performance liquid chromatography (HPLC) The results of the study are shown in Table 3.15
Table 3.15: Isolated compounds from fruits of Melia azedarach
3 MA3 Quercetin 3-O-[
-L-rhamnopyranosyl-(1→6)]-glucopyranoside
3.1.2 Structural elucidation of new compound MA1
Compound MA1 was isolated as optically active colorless syrup and the HRESIMS analytical data of 1 revealed the molecular formula as C37H44O9 (m/z 655.2879, [M+Na]+) The UV absorption maxima at 205, 218, 244, and 273 nm indicated the presence of a conjugated benzene chromophore [99] The 1H-NMR
spectrum of MA1 (Table 3.2) displayed the characteristic resonances for four
methyl singlets [H 1.00 (6H, CH3-20, -22), 1.18 (3H, CH3-18), and 1.25 (3H,
CH3-21)], one oxymethylene group [H 3.13 (m) and 3.45 (d, J = 7.5 Hz)], and
one set of signals for furan moiety [H 6.16 (br s), 7.13 (br s), and 7.28 (br s)],
respectively, which indicated the basic skeleton of MA1 was similar to that of
trichilinins [48] In addition, there were also proton signals representative for two acetyl singlets [H 1.90 (3H) and 1.91 (3H)] and one benzoyl fragment [H 7.43 (2H, t, 7.5), 7.56 (1H, t, 7.5), and 8.08 (2H, d, 7.5)] Comparison of the above spectral data with those of trichilinin E [48], the observed difference in 1 was the
presence of one more acetyl group The substitution position of acetyl and benzoyl groups were determined by the assistance of comprehensive 2D NMR
spectroscopic examinations of 1 In the HMBC spectrum, 2J, 3J-HMBC
correlations from 3 to C-1, C-27; from 6 to C-7; from 7 to C-1'; from
H-12 to C-29; from H-15 to C-8, C-17; from H-17 to C-23, C-24, C-26; from CH3
Trang 12-18 to C-3, C-4, C-19; from CH3-20 to C-1, C-5, C-9; from CH3-21 to C-14; from
CH3-22 to C-12, C-14, C-17; from H-7' to C-1', respectively, were observed and established its 2D planar structure as shown (Figure 1) The other successive 2D
experiments constructed the same stereochemical configurations of 1 and
completed the assignments of all the proton and carbon signals Conclusively, the
chemical structure of 1 was established as
3α,12α-diacetoxy-7α-benzoyloxy-1α-hydroxytrichilinin (Figure 1) and named trivially as trichilinin F followed the previous convention
Table 3.2: 1 H and 13 C-NMR data of MA1